Anti-cancer nucleosides are grouped as antimetabolites, hypomethylating agents or others depending on their mechanism of action. They are typically analogs of natural nucleosides of cytidine, adenosine, guanosine and thymidine (B. Ewald, et al., Oncogene, 2008, 27:6522-6537).
For example, cytarabine (hereinafter, referred to as “Ara-C”) is a cytosine nucleoside analog and inhibitor of DNA polymerase. It can prevent DNA synthesis, be also incorporated into the DNA and interfere with DNA replication. Currently, Ara-C is mainly used for the treatment of acute leukemia. However, Ara-C has no effect on the majority of solid tumors. This is due to the fact that solid tumors have high level of cytidine deaminase converting the Ara-C into inactive ara-uridine (T. Ohta, et al., Oncology Reports, 2004, 12:1115-1120). Oral absorption rate of Ara-C is low, less than 20%, and clearance rate is high, which prohibits the oral dosing (O. Schiavon, et al., European Journal of Medicinal Chemistry, 2004, 39:123-133). Instead, it is prescribed as continuous intravenous infusion. Many different types of prodrugs and derivatives have been developed and tested clinically (A. Hamada, et al., Clinical pharmacokinetics, 2002, 41:705-716). Lipid-modified Ara-C, called elacytarabine, showed much improved preclinical efficacy in terms of delivery and potency (A. C. Burke, et al., Expert Opinion on Investigational Drugs, 2011, 20:1707-1715). However, it failed to show the clinical efficacy. A cytidine deaminase-resistant analog BCH-4556 (H. Gourdeau, et al., Cancer Chemotherapy and Pharmacology, 2001, 47:236-240) showed a higher potency than Ara-C, but failed to show the desired efficacy in clinical study.
In order to improve the efficacy of Ara-C, patients are generally treated with Ara-C combined with other drugs, such as, daunorubicin, all-trans retinoic acid, arsenic trioxide, pirarubicin, etoposide, cyclophosphamide and fludarabine. Ara-C has side effects such as bone marrow suppression, gastrointestinal side reaction. A small number of patients may have abnormal liver function, fever, rash and other side effects (J. M. Bennett, Leukemia Research, 2003, 27:761; and H. M. Kantarjian, Cancer, 2007, 109:1007-1010).
Zebularine is known as a DNA methyltransferase (DNMT) inhibitor, hypomethylating agent, or a demethylating agent. DNA methyltransferases transfer methyl groups from the methyl donor S-adenosyl methionine (SAM) to cytosine of a CpG islands on DNA molecule. Several biological functions for the methylated bases in DNA, including regulation in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes have been reported (Razin and Riggs, eds. in DNA Methylation Biochemistry and Biological Significance, Springer-Verlag, New York, 1984).
Short chain fatty acids including butyric acid, isobutyric acid, valeric acid, propionic acid, phenylbutyric acid, and its derivative valproic acid are known to be histone deacetylases inhibitors (HDACIs), and affect cell proliferation, gene expression and differentiation.
Histone deacetylases (HDACs) are a group of enzymes responsible for deacetylation of histones and nonhistone proteins. Lysine acetylation, i.e., the transfer of an acetyl moiety from acetyl-coenzyme A to the ε-amino group of a specific lysine residue, is one of the major forms of post-translational modifications of histones, and has been correlated with transcription, chromatin assembly, and DNA repair (Marks et al., Nat Rev Cancer, 1:194-202, 2001).
Aberrant acetylation of histone tails, emerging from either HAT (histone acetyl transferases) mutation or abnormal recruitment of HDACs, is known to be associated with carcinogenesis (P. P. Pandolfi, Oncogene, 20: 3116-3127, 2001). In various cases, altered HAT or HDAC activity has been identified in a variety of cancers. HDAC inhibitors (HDACIs) are potent inducers of histone acetylation, cell growth arrest, and differentiation and apoptosis of several cell lines. They constitute a novel class of chemotherapeutic agents initially identified by their ability to reverse the malignant phenotype of transformed cells. They activate differentiation programs, inhibit cell cycle, and induce apoptosis in a wide range of tumor-derived cell lines, and thereby blocking angiogenesis and stimulating the immune system in vivo (P. A. Marks et al., Nat Rev Cancer 2001, 1:194-202; and R. W. Johnstone, Nat Rev Drug Discovery 2002, 1:287-299).
Combination therapy could give an additive or synergistic effect on anti-cancer treatment in various trials. In particular, combinations of DNA hypomethylating agents with histone deacetylase inhibitors were effective on various cancers. For example, DAC is a widely used anti-leukemic agent, and its effects were greatly improved when combined with valproic acid tested on MOLT-4 and HL-60 leukemic cell lines (H. Yang, et al., Leukemia Research, 29, 739-748, 2005). The synergistic effect was correlated with DNA demethylation and histone acetylation and with reactivation of the tumor suppressor genes p21CIP and p57KIP2.
A mutual prodrug (hybrid drug, MP) consists of two pharmacologically active agents coupled together so that each acts as a promoiety for the other agent and vice versa. Constituent drugs can be covalently coupled either directly or via a suitable linker (A. K. Jain, et al., Bioorganic Chemistry, 2013, 49:40-48). Mutual prodrugs are particularly useful for two or more constituents with potential synergistic or additive effects but difficult to be formulated in a combination pill. When two synergistic agents are administered individually but simultaneously, they will be transported to the site of action with different efficiencies. However, it is desirable to have the two agents reach a site simultaneously. The mutual prodrug strategy may be used to resolve the problem mentioned above.
Accordingly, there has been a continued need to develop a novel mutual prodrug compound that has improved growth inhibitory activity and exhibits pharmacokinetic properties of drug delivery.